Using NMR spectrometry and reporter assays we identified liver receptor homolog 1 (LRH-1) transcriptional antagonists. We hypothesize that LRH-1-selective antagonists would be useful for treating or preventing recurrence of estrogen-dependent breast cancer in postmenopausal women. We base this hypothesis on the following findings: After menopause, estrogen is produced locally in breast tissue rather than coming from the ovary. In the postmenopausal cancerous breast, expression of the enzyme synthesizing estrogen (aromatase) becomes elevated, as do estrogen and the estrogen nuclear receptor (ER). Moreover, compared to other tissues, aromatase is expressed by activation of a different promoter that is responsive to binding by LRH-1, an orphan nuclear receptor. LRH-1 is also overexpressed in the postmenopausal cancerous breast. There, the major producer of estrogen is the preadipocyte followed by the cancer cell. Thus, treatment with an LRH-1 antagonist would locally reduce estrogen levels compared to adjuvant therapeutics such as estrogen antagonists and aromatase inhibitors and, thereby, attenuate the adverse systemic effects caused by whole-body estrogen ablation. Use of a dual therapeutic approach may also extend the treatment period for adjuvant therapy until relapse occurs. LRH-1 also activates the ER gene promoter to induce the expression of the ER, which in the presence of estrogen is activated to drive breast cancer cell proliferation. LRH-1 also has independent roles in cell proliferation and invasion that are related to its ability to induce the expression of other genes such as cyclin D1 and c-Myc. Therefore, treatment with an LRH-1 antagonist could block estrogen-dependent breast cancer cell proliferation and invasion. We propose a three-year multidisciplinary project involving three Specific Aims: (1) LRH-1 antagonist design and synthesis based on direct design and SAR approaches. The lead antagonist was first found to interact with another orphan nuclear receptor, small heterodimer partner. On the basis of computational analysis, we hypothesize that appropriate scaffold substitutions will selectively enhance interaction with the LRH-1 ligand-binding domain and improve antagonism of transcriptional activity. (2) Bioevaluation in preadipocyte and estrogen-dependent cell lines coupled with mutagenesis studies will enable us to identify an LRH-1 pharmacophore and ligand-binding pose in the human LRH-1 ligand-binding pocket to optimize activity. The antagonists will also permit us to investigate LRH-1 signaling pathways in breast cancer cells and preadipocytes. (3) In vivo validation in an estrogen-dependent breast cancer xenograft model in ovariectomized immunodeficient mice inoculated with engineered cancer cells stably expressing LRH-1 and treated with the estrogen precursor androstenedione.